Estimation of HDL Cholesterol 

 

Introduction

• HDL stands for High-Density Lipoprotein, commonly called good cholesterol.
• HDL is one of the major classes of plasma lipoproteins.
• It is synthesized mainly in the liver and intestine.
• HDL contains apolipoproteins, phospholipids, cholesterol, and proteins.
• Its main function is to remove excess cholesterol from peripheral tissues and transport it back to the liver.
• This process is called reverse cholesterol transport.
• In the liver, cholesterol is converted into bile acids and excreted through intestine.
• HDL therefore protects blood vessels against cholesterol deposition.
• Serum HDL estimation is very important in cardiovascular risk assessment.
• Low HDL level increases risk of coronary heart disease.

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Principle

• HDL cholesterol estimation is based on direct enzymatic selective method.
• PVS and PEGME block LDL, VLDL, and chylomicrons, so only HDL reacts.
• HDL cholesterol is hydrolyzed by cholesterol esterase to free cholesterol.

Reaction 1

HDL Cholesterol Ester + H₂O → Cholesterol + Fatty acids

• Free cholesterol is oxidized by cholesterol oxidase to produce hydrogen peroxide.

Reaction 2

Cholesterol + O₂ → H₂O₂ + Cholestenone

• Hydrogen peroxide reacts with 4-aminoantipyrine + TODB in presence of peroxidase to form colored quinoneimine dye.

Reaction 3

2H₂O₂ + 4-AA + TODB → Quinoneimine dye + 5H₂O

• Color intensity is directly proportional to HDL concentration.
• Absorbance is measured at 600 / 700 nm.

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Specimen

Sample Type

• Serum is preferred specimen
• Heparin plasma can also be used

Precautions

• Use fresh non-hemolyzed sample
• Avoid contaminated sample

Stability

• 24 hours at 20–25°C
• 7 days at 4–8°C
• 12 weeks at −20°C

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Reagents

Reagent 1 (R1)

• MES buffer (pH 6.5)
• N,N-Bis(4-sulfobutyl)-3-methylaniline
• Polyvinyl sulfonic acid
• Polyethylene glycol methyl ester
• Magnesium chloride

Reagent 2 (R2)

• MES buffer
• Cholesterol esterase
• Cholesterol oxidase
• Peroxidase
• 4-aminoantipyrine
• Detergent

Reagent Preparation

• Both reagents are liquid and ready to use

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Materials Required

• Test tubes
• Micropipette
• Pipette tips
• Colorimeter / semi-auto analyzer
• Cuvette
• Timer
• HDL reagent kit

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Procedure

First Incubation

• Mix properly
• Incubate at 37°C for 5 minutes

Then Add

Second Incubation

• Mix properly
• Incubate at 37°C for 5 minutes

Reading

• Read final absorbance against reagent blank
• Measure at 600 / 700 nm

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Calculation

Formula

HDL-C = (Absorbance of Sample / Absorbance of Calibrator) × Concentration of Calibrator

Normal Reference Values

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Clinical Significance

Decreased HDL

• Coronary heart disease risk
• Atherosclerosis
• Diabetes mellitus
• Obesity
• Smoking

Increased HDL

• Protective against cardiovascular disease
• Good lipid metabolism

Diagnostic Importance

• Assesses cardiovascular protection
• Evaluates lipid profile
• Helps estimate cardiac risk